Abstract
Electron cryotomography is the highest-resolution structural technique currently available that can be applied to unique objects such as flexible large protein complexes, irregular viruses, organelles and small cells. Specimens are preserved in a near-native, 'frozen-hydrated' state by vitrification. The thickness of the vitreous ice must be optimized for each specimen, and gold fiducials are typically added to facilitate image alignment. Here, we describe in detail our protocols for electron cryotomography sample preparation including (i) introduction of fiducial markers into the sample and (ii) sample vitrification. Because we almost exclusively use an automated, climate-controlled plunge-freezing device (the FEI Vitrobot) to vitrify our samples, we discuss its operation and parameters in detail. A session in which eight grids are prepared takes 1.5–2 h.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Rent or buy this article
Prices vary by article type
from$1.95
to$39.95
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Lucic, V., Forster, F. & Baumeister, W. Structural studies by electron tomography: from cells to molecules. Annu. Rev. Biochem. 74, 833–865 (2005).
Nickell, S., Kofler, C., Leis, A.P. & Baumeister, W. A visual approach to proteomics. Nat. Rev. Mol. Cell Biol. 7, 225–230 (2006).
Adrian, M. et al. Direct visualization of supercoiled DNA molecules in solution. EMBO J. 9, 4551–4554 (1990).
Li, H., DeRosier, D.J., Nicholson, W.V., Nogales, E. & Downing, K.H. Microtubule structure at 8 Å resolution. Structure 10, 1317–1328 (2002).
Angell, C.A. Amorphous water. Annu. Rev. Phys. Chem. 55, 559–583 (2004).
Dubochet, J. & McDowall, A.W. Vitrification of pure water for electron microscopy. J. Microsc. 124, RP3–4 (1981).
Lepault, J., Booy, F.P. & Dubochet, J. Electron microscopy of frozen biological suspensions. J. Microsc. 129, 129–102 (1983).
Adrian, M., Dubochet, J., Lepault, J. & McDowall, A.W. Cryo-electron microscopy of viruses. Nature 308, 32–36 (1984).
Frederik, P.M. & Hubert, D.H. Cryoelectron microscopy of liposomes. Meth. Enzymol. 391, 431–448 (2005).
Murphy, G.E. & Jensen, G.J. Electron cryotomography of the E. coli pyruvate and 2-oxoglutarate dehydrogenase complexes. Structure 13, 1765–1773 (2005).
Iancu, C.V., Wright, E.R., Heymann, J.B. & Jensen, G.J. A comparison of liquid nitrogen and liquid helium as cryogens for electron cryotomography. J. Struct. Biol. 153, 231–240 (2006).
Wright, E.R., Iancu, C.V., Tivol, F.W. & Jensen, G.J. Observations on the behavior of vitreous ice at approximately 82 and approximately 12 K. J. Struct. Biol. 153, 241–252 (2006).
Benjamin, J., Ganser-Pornillos, B.K., Tivol, W.F., Sundquist, W.I. & Jensen, G.J. Three-dimensional structure of HIV-1 virus-like particles by electron cryotomography. J. Mol. Biol. 346, 577–588 (2005).
Iancu, C.V. et al. A 'flip-flop' rotation stage for routine dual-axis electron cryotomography. J. Struct. Biol. 151, 288–297 (2005).
Komeili, A., Li, Z., Newman, D.K. & Jensen, G.J. Magnetosomes are cell membrane invaginations organized by the actin-like protein MamK. Science 311, 242–245 (2006).
Henderson, G.P. & Jensen, G.J. Three-dimensional structure of Mycoplasma pneumoniae's attachment organelle and a model for its role in gliding motility. Mol. Microbiol. 60, 376–385 (2006).
Murphy, G.E., Leadbetter, J.R. & Jensen, G.J. In situ structure of the complete Treponema primitia flagellar motor. Nature 442, 1062–1064 (2006).
Briegel, A. et al. Multiple large filament bundles observed in Caulobacter crescentus by electron cryotomography. Mol. Microbiol. 62, 5–14 (2006).
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing financial interests.
Rights and permissions
About this article
Cite this article
Iancu, C., Tivol, W., Schooler, J. et al. Electron cryotomography sample preparation using the Vitrobot. Nat Protoc 1, 2813–2819 (2006). https://doi.org/10.1038/nprot.2006.432
Published:
Issue Date:
DOI: https://doi.org/10.1038/nprot.2006.432
This article is cited by
-
Stepwise assembly and release of Tc toxins from Yersinia entomophaga
Nature Microbiology (2024)
-
L-form conversion in Gram-positive bacteria enables escape from phage infection
Nature Microbiology (2023)
-
Actin cytoskeleton and complex cell architecture in an Asgard archaeon
Nature (2023)
-
Rhoptry secretion system structure and priming in Plasmodium falciparum revealed using in situ cryo-electron tomography
Nature Microbiology (2022)
-
Structure of a thylakoid-anchored contractile injection system in multicellular cyanobacteria
Nature Microbiology (2022)
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.